The invention relates in general to an apparatus and method for achieving electrophoretic focusing, and in particular to an apparatus for achieving electrophoretic separation and purification which is characterized by a separation chamber formed between two precision-pore, insulated screens and which also includes inlet and outlet ports, a plurality of purge chambers for extracting extraneous fractions and for providing thermal cooling, a plurality of electrodes to provide a transverse electric field in the separation chamber, and pumping means for pumping sample, carrier buffer, purge buffer and electrode rinse buffer through the apparatus, and a method of employing this apparatus to achieve separation and collection of a desired component from a biological or chemical sample.
There are two electrokinetic methods that have had success separating biological materials, namely, zone electrophoresis and isoelectric focusing. Electrophoresis is the movement of suspended or dissolved charged particles in response to an applied electric field. The rate of motion depends upon the charge, size and shape of the particles and specific properties of the solvent buffer and its container. In zone electrophoresis, the components in a short sample zone are separated by the action of the electric field. The injection of a narrow, uniform zone and the absence of dispersive fluid flows are necessary conditions for successful operation. Significant sources of dispersion are: 1) uneven (parabolic) flows; 2) electrohydrodynamic flows; 3) molecular diffusion; 4) thermal convection; 5) sedimentation; 6) thermally induced sample mobility variations; and 7) electroosmosis.
In continuous zone electrophoresis (CFE), the electrolyte solution flows in a direction perpendicular to the electric field and the mixture to be separated is inserted continuously into the flowing solution. Components of the mixture are deflected according to their electrophoretic mobilities and can be collected continuously in a finite array of collection ports after their migration. Svensson and Braftsten were the first to report a method for carrying out electrophoresis continuously. They used a lateral electric field in a narrow Plexiglas box packed with glass powder as an anti-convective medium. Durrum modified the above configuration by replacing the glass-filled box with a filter paper curtain, hanging in a free vapor space. While both of these methods demonstrated continuous electrophoresis, they both used a stabilizing medium. Anti-convective media cause many problems such as reduction of the flow capacity by their presence, electroosmosis in the interstices, adsorption of the sample and xe2x80x9cpacking or eddy diffusionxe2x80x9d. Efforts were then made to do continuous electrophoresis in a free fluid. Dobry and Finn (U.S. Pat. No. 3,149,060) were the first to report continuous flow free fluid electrophoresis in a rectangular chamber with a cross-section of low aspect ratio, hence providing little resistance to thermal convective flow disturbances. This configuration was limited to very low electric fields and required the use of buffer thickening agents to suppress convective eddies. Philpot described a continuous flow electrophoresis system with the electric field applied across (perpendicular to) a thin film of liquid. He later wrapped his thin film geometry into a thin annulus surrounded by two concentric cylinders (electrodes). The outer cylinder rotated to provide a stabilizing velocity gradient.
Mel in 1959 reported the first use of a high aspect ratio rectangular separation chamber using a lateral electric field. The xe2x80x9cthinxe2x80x9d chamber of 0.7 cm thickness provided the necessary wall interaction to suppress thermal convective flows to the extent that a less viscous free flow buffer could be used. This design served as the impetus for the development of the conventional CFE machines of the 60""s and 70""s with their chamber cross-section of high aspect ratio and laterally directed electric fields. During this time frame, Hannig and his co-workers developed CFE by making the chamber cross-sections even thinner, approaching 0.25 cm for some designs. Unfortunately, the gains made in suppressing thermal convection were wiped out by electrohydrodynamic interaction with intrinsic chamber fluid flows to cause crescent-shaped distortions. Nevertheless, a variety of CFE instruments were manufactured according to the designs of Hannig (in Germany) and Strickler (in the US) (U.S. Pat. No. 3,412,008) and several hundred instruments were used in laboratories around the world. Rhodes and Snyder subsequently devised a technique to minimize these flow distortions (U.S. Pat. No. 4,752,372).
While the concept of using a counter flow to oppose the electrophoretic migration velocity has long been considered an attractive means to achieve a focusing effect, no method has been found to provide the uniform velocity field necessary to bring this concept to fruition. Richman patented a counter-flow method where axial bands of electroosmotic coatings of varying zeta potential would xe2x80x9cstraightenxe2x80x9d distorted sample bands (U.S. Pat. No. 4,309,268). The method was impractical because most coatings change with time and there exists no spectrum of coatings with respect to zeta potential. A more practical approach that did not use counter-flow was suggested by Strickler wherein the CFE was divided into two vertical compartments, each with a different wall coating, so that the combined electroosmotic flow would yield a more coherent sample band. Subsequently, Ivory used counter-flow to increase sample residence time in a recycling CFE. Egen, et al. have also devised a counterflow gradient focusing method (U.S. Pat. No. 5,336,387).
While the crescent phenomenon was long known to cause untenable sample stream distortion in CFE instruments, it was not until 1989 that Rhodes and Snyder showed that electrohydrodynamics transforms initially circular sample streams into ribbons that initiate the crescent shaped distortions. The operation of CFE devices was labor intensive and unreliable due to contamination of the closely spaced chamber walls and the resultant electroosmotic flow variations through the chamber.
Isoelectric focusing (IEF) is an electrophoretic technique that adds a pH gradient to the buffer solution and together with the electric field focuses most biological materials that are amphoteric. Amphoteric biomaterials such as proteins, peptides, nucleic acids, viruses, and some living cells are positively charged in acidic media and negatively charged in basic media. During IEF, these materials migrate in the pre-established pH gradient to their isoelectric point where they have no net charge and form stable, narrow zones. In spite of the very long time required for isoelectric focusing, this process yields high resolution bands because any amphoteric biomaterial which moves away from its isoelectric point due to diffusion or fluid movement will be returned by the combined action of the pH gradient and electric field. The focusing process thus purifies and concentrates sample into bands that are relatively stable. This is a powerful concept that has yielded some of the highest resolution separations, especially when coupled with electrophoresis in two-dimensional gels.
IEF had its practical beginning in the mid-1950""s when Kolin first demonstrated the concept of focusing ions in a pH gradient by placing a molecular sample between an acidic and a basic buffer and applying an electric field. Although the constituents focused rapidly, the gradient soon deteriorated due to the concurrent electrophoretic migration of all of the buffering ions. The synthesis of stable carrier ampholytes by Vesterberg and their successful commercial development led to broad use in gels or other restrictive media to suppress electroosmosis and thermal convection during analytical separations.
The high resolution achieved by IEF encouraged many attempts to develop a preparative version of the process. This proved to be much more difficult for IEF than zone electrophoresis because of the variable fluid properties and sample characteristics within the chamber leading to changing values of electroosmosis and thermal convection during the separation. Various CFE devices were modified to run with an amphoteric mixture instead of buffer but the problems (long focusing time requiring a slow flow through the chamber, pH drift toward the cathode, reduced voltage/current levels for acceptable heating and convection) became insurmountable. Bier developed an external cooling system, added sensors and demonstrated the improved focusing with recycling (U.S. Pat. No. 4,362,612). Bier then added a stabilizing assembly rotation to the membrane segmentation and a novel collection system (U.S. Pat No. 4,588,492) which led to the Roto-Phor from Bio-Rad (Hercules, Calif.).
Unfortunately there are drawbacks to IEF that have limited its applications. The rate of electrophoretic migration of each charged species decreases progressively as it approaches its isoelectric point and long residence times are required for high resolution. Proteins have reduced solubility at their isoelectric point although precipitation of the concentrated bands can be minimized by addition of detergent. Additional problems relate to the commercial amphoteric solutions, including: 1) difficulty of extracting the separated proteins, peptides, etc., from the amphoteric solutions because of their similar physical properties and interactions; 2) chemical toxicity; 3) handling problems; and 4) cost. IEF has also been hindered by problems during the transition from an analytical system to a preparative system that have limited its intended use. It is thus highly desirable to develop a focusing system for separating biological molecules and other components in a mixture which is able to avoid all of the problems of the prior art and which can achieve high resolution of separation in an analytical or a preparative mode through a practically unlimited scale-up potential. It is also highly desirable to develop an electrophoretic focusing system which can control the adverse effects of Joule heating and electrohydrodynamics on the electrophoretic separation procedure.
It is an object of the present invention to provide a preparative-scale free-fluid electrophoretic separator with high resolution as well as an analytical capability commensurate with capillary zone electrophoresis. The particular mode of high-resolution separation as provided by the present invention, which is referred to as electrophoretic focusing, combines features of electrophoresis and isoelectric focusing to accomplish large scale purifications and fractionations that have not been possible before now.
Many research and applications tasks with biological materials require a large source of highly purified biologically active molecules. The diverse supply of materials for biotechnology ranging from plants to genetically derived sources are placing increased demands on separation and purification. Existing preparative separation techniques yield products with a variety of impurities that can be measured analytically but not removed. Analytical techniques have been perfected in recent years but attempts to scale these techniques into larger production have relied on generally increasing the physical dimensions instead of investigating a new technique. It is an advantage of the focusing device of the present invention that it will be able to purify biological materials in amounts and to purity levels above those now obtainable.
In accordance with the present invention, there is provided an electrophoretic focusing apparatus and method which is useful in achieving the separation and purification of particular components of a mixture of biological or chemical materials. The general purpose of the invention is a continuous processing system that separates and purifies any soluble or microparticulate sample that acquires a surface electric charge when immersed in a polar (e.g. aqueous) fluid environment. It combines the best features of electrophoresis and isoelectric focusing in a novel device that incorporates a combination of transverse electric field and buffer flow field to focus and collect any selected biological component. Although the high resolution achievable by focusing is familiar to isoelectric focusing, electrophoretic focusing avoids many of its problems, such as the need for complex buffers and the long times required for the molecules to reach their isoelectric point. This new concept incorporates a large-gap chamber and control of all sources of sample dispersion. The design of the electrophoretic focusing chamber combined with the orientation and magnitude of the electric fields and buffer flows are planned to eliminate sample dispersion. The large gap will keep sample away from the walls as well as increase its throughput.
It is another object of the present invention to develop a separation device capable of high speed and short residency through the use of high voltage gradients. These high voltage gradients are produced by relatively low voltages applied across the narrow chamber dimensions. The goal of high resolution of separation can be achieved through the use of the present invention in an analytical or a preparative mode through a practically unlimited scale-up potential. A further goal is to control the adverse effects of Joule heating and electrohydrodynamics.
These and other objects and benefits are achieved by the use of the present invention which provides a number of innovations and insights with regard to fundamental fluid and thermal geometries and operations. The focusing is accomplished with a minimum of sample migration which leads to a higher resolution in a shorter time. Adiabatic thermal conditions in the lateral (scale-up) dimension permit a large increase in throughput at no apparent loss of resolution. Active cooling limits the maximum chamber temperature and its relationship to the chamber orientation and buffer fluid transport is such as to limit thermal convection. Porous, rigid screens permit a controlled focusing cross-flow which balances the electrophoretically-driven sample velocity. In the preferred method in accordance with the invention, separation and collection of at least one component from a mixture of components is obtained by the steps of (a) providing an apparatus comprising a separation chamber and a plurality of purge chambers, and establishing a first buffer flow in the separation chamber in the axial direction, said first buffer flow having a first flow rate; (b) establishing a second buffer flow in the separation chamber consisting of two flows on either side of the first flow that converge on the first flow at the chamber entrance and diverge from the first flow at the chamber exit; (c) establishing a third buffer flow in each of at least two purge chambers in the axial direction, said second buffer flow having a second flow rate, said second buffer fluid flow having a second flow rate higher than that of the first flow rate; (d) introducing two precision-pore screens that partition the said separation chamber from each of the two said purge chambers; (e) establishing a fourth buffer flow by the biasing of the purge valves to control said fourth buffer flow from one of the purge chambers through a precision-pore screen transversely into the separation chamber, then out of the separation chamber through the second precision-pore screen into a second purge chamber, thus providing the required uniform focusing fluid velocity in the separation chamber; (f) introducing the mixture of sample components with the said first buffer flow directly into the separation chamber flow entrance or through at least one injection port located in the separation chamber interior; (g) controlling the second buffer flow to converge and thin the first buffer flow with sample components at the separation chamber entrance and then diverge and extract sample components at the separation chamber exit; and (h) applying an electrical potential transversely across the separation chamber in the form of a constant voltage gradient to impart electrophoretic velocity to the fractional components in the separation chamber in the transverse direction perpendicular to the first buffer flow direction and parallel to the fourth buffer flow direction.